Simultaneous sequencing of nucleic acids

ABSTRACT

The invention concerns a method for the sequence-specific labelling of nucleic acids comprising the generation of labelled nucleic acid fragments by an enzymatic labelling reaction in which a labelled deoxyribonucleoside triphosphate is attached to a nucleic acid primer molecule and the nucleic acid sequence is determined by means of the label, wherein the labelling reaction is carried out in a single reaction vessel with the simultaneous presence of one or several nucleic acid primer molecules and at least two labelled deoxyribonucleoside triphosphates which each contain different labelling groups and different bases and under conditions in which only one single type of labelled deoxyribonucleoside triphosphate can be attached to a nucleic acid primer molecule.

The present invention concerns a method for the sequence-specificlabelling and simultaneous sequencing of nucleic acids comprising theproduction of labelled nucleic acid fragments by an enzymatic labellingreaction in which a labelled deoxyribonucleoside triphosphate isattached to a nucleic acid primer molecule and the nucleic sequence isdetermined by means of the label.

In DNA sequencing by the enzymatic chain termination method according toSanger one starts with a nucleic acid template from which many labellednucleic acid fragments of various length are produced by an enzymaticextension and termination reaction in which a synthetic oligonucleotideprimer is extended and terminated with the aid of polymerase and amixture of deoxyribonucleoside triphosphates (dNTP) and chaintermination molecules, in particular dideoxyribonucleoside triphosphates(ddNTP).

In this method a mixture of the deoxyribonucleoside triphosphates(dNTPs) and one dideoxyribonucleoside triphosphate (ddNTP) is used ineach of four reaction mixtures. In this manner a statisticalincorporation of the chain termination molecules into the growingnucleic acid chains is achieved and after incorporation of a chaintermination molecule the DNA chain cannot be extended further due to theabsence of a free 3'-OH group. Hence numerous DNA fragments of variouslength are formed which, from a statistical point of view, contain achain termination molecule at each potential incorporation site and endat this position. These four reaction mixtures which each containfragments ending at a base due to the incorporation of chain terminationmolecules are separated according to their length for example bypolyacrylamide gel electrophoresis and usually in four different lanesand the sequence is determined by means of the labelling of thesenucleic acid fragments.

Nowadays DNA sequencing is carried out with automated systems in whichusually a non-radioactive label, in particular a fluorescent label, isused (L. M. Smith et al, Nature 321 (1986), 674-679; W. Ansorge et al,J. Biochem. Biophys. Meth. 13 (1986), 315-323). In these automatedsystems the nucleotide sequence is read directly during the separationof the labelled fragments and entered directly into a computer.

In the automated methods for sequencing nucleic acids non-radioactivelabelling groups can either be introduced by means of labelled primermolecules, labelled chain termination molecules or as an internal labelvia labelled dNTP. In all these known labelling methods the sequencingreactions are in each case carried out individually in a reaction vesselso that always only one single sequence is obtained with a sequencingreaction.

A method for sequencing nucleic acids is described in the InternationalPatent Application WO 93/03180 in which nucleic acid fragments aredetermined after incorporation of at least one non-radioactivelylabelled dNTP as an internal label wherein the internal label isincorporated into the nucleic acid fragments in the absence of chaintermination molecules.

The simultaneous determination of two nucleic acid sequences using twodifferently labelled primer molecules in a single reaction vessel wasdescribed by Wiemann et al (Anal. Biochem. 224 (1995) 117-121). However,a disadvantage of this method is that dyelabelled oligonucleotideprimers have to be used which are expensive or/and complicated toproduce. This is a particular disadvantage when sequencing longer genesections using the walking primer strategy in which different primershave to be used for each sequencing reaction. Moreover the use oflabelled primers sometimes impedes the hybridization of the primer withthe nucleic acid template which can lead to inaccurate sequencingresults.

In order to avoid this disadvantage one could carry out two sequencingreactions each with an unlabelled primer and one of two differentlylabelled dNTP in two separate reaction vessels, mix both products, applythem simultaneously to a gel and determine the sequences. However, adisadvantage of this method is that it is laborious and complicated tocarry out two sequencing reactions in separate reaction vessels.

Thus the object of the present invention was to provide a method for thesequence-specific labelling and sequencing of nucleic acids in which thedisadvantages of the state of the art are at least partially eliminated.In particular the method according to the invention should enable two ormore sequencing reactions to be carried out simultaneously in a singlereaction vessel using at least two differently labelled dNTP and atleast two preferably unlabelled primer molecules.

The object of the invention is achieved by a method for thesequence-specific labelling of nucleic acids comprising the productionof labelled nucleic acid fragments by an enzymatic labelling reaction inwhich a labelled deoxyribonucleoside triphosphate is attached to anucleic acid primer molecule and the nucleic acid sequence is determinedby means of the label, the method being characterized in that thelabelling reaction takes place in a single reaction vessel with thesimultaneous presence of one or several nucleic acid primer moleculesand at least two labelled deoxyribonucleoside triphosphates which eachcontain different labelling groups and different bases and underconditions in which only one single type of labelled deoxyribonucleosidetriphosphate can be attached in each case to a nucleic acid primermolecule.

The nucleic acid primer molecule is a nucleic acid, preferably a DNA,which is sufficiently complementary to a nucleic acid present in thereaction mixture to be able to hybridize with it under the respectivereaction conditions. The length of the primer molecule is preferably10-100, particularly preferably 12-50 and most preferably 12-30nucleotides.

The enzymatic labelling reaction is carried out in the method accordingto the invention such that only one single type of labelled dNTP andpreferably only one single type of labelled dNTP and preferably only onesingle labelled dNTP is attached in each case to a nucleic acid primermolecule. The labelled dNTP is preferably attached directly to the 3'end of the primer (position +1). In this case the base of the labelleddNTP is complementary to that base which is located on the nucleic acidhybridizing with the primer molecule directly on the 5' side of thecomplementary region to the primer. If it can be ensured that only onesingle type of label is incorporated behind each primer, the label canalso be incorporated at a greater distance from the 3' end of the primer(positions +2, +3, +4 etc.). In addition to the at least two differentlylabelled dNTPs, unlabelled dNTPs may also be present in the labellingreaction.

However, the sequence of one or several primer molecules can also beselected such that, when using a polymerase with 3' exonucleaseactivity, the labelled dNTPs are inserted as a terminal base of theprimer or primers in an exchange reaction when the 3' terminal base ofthe primer is the same as the base used for the labelled dNTP.

When carrying out the enzymatic labelling reaction the conditions arechosen such that statistically only one single type of labelleddeoxyribonucleoside triphosphate can be attached to the primer. In orderto achieve this reaction specificity the reaction conditions (such asthe concentration of the polymerase enzyme or/and dNTP, temperature,concentration of other components etc.) are preferably selectedaccordingly. In addition the specificity of the reaction can beincreased by selection of the primer molecules as explained in thefollowing.

The method according to the invention enables for example thesimultaneous sequencing of DNA fragments with two or several primermolecules, preferably unlabelled primer molecules, and several labelleddNTPs in one reaction vessel. Several labelling reactions can be carriedsimultaneously on different strands of the nucleic acid to be sequencedor at various sites on the same nucleic acid strand. The specificity ofthe method according to the invention is surprisingly so high thatstatistically a single type of label, in particular only one singlelabelled dNTP is attached to a particular primer molecule even ifseveral other differently labelled dNTP, primer molecules and nucleicacid template molecules are present in the same reaction vessel.

The combination of one or several DNA templates and of two or severalprimer molecules and labelled dNTPs in a single sequencing reactionenables the costs for carrying out the sequencing reactions to bereduced by 50-75%. There is a reduction in the costs for the chemicalsas well as for the amount of work required. This is of particularimportance for large scale sequencing projects such as the human genomesequencing project or other genome projects.

There is a wide range of application forms for the method according tothe invention e.g. sequencing, mapping and selective labelling of DNAfragments in medical diagnostics, molecular biology, biochemistry andchemistry. The most important form of application is automated DNAsequencing with fluorescent marker groups.

Specific forms of application are the so-called mini-sequencing in whichthe nucleic acid sequence is only determined within a limited region ofup to three bases after the primer based on a sequence-specificincorporation of the labelled dNTP. A further possible application is todetect point mutations in genes, in particular in human genes. In thiscase the primer molecule is selected such that it lies immediately infront of a hot spot of the mutation. Only one primer is used togetherwith two or several differently labelled dNTP and the labelling reactionis carried out. Only a single labelled dNTP is incorporated which iscomplementary to the corresponding base on the nucleic acid template.Subsequently the extended primer molecule can be analysed and thelabelling group can be identified by its spectral characteristics. Inthis manner it is possible to draw conclusions about the sequence of therespective gene.

When used to determine the sequence of nucleic acids, the methodaccording to the invention is preferably carried out by

a) binding at least one nucleic acid primer molecule to the nucleic acidto be sequenced,

b) adding a polymerase and at least two labelled deoxyribonucleosidetriphosphates which each contain different labelling groups anddifferent bases,

c) incubating the reaction mixture in a single reaction vessel undersuch conditions that in each case only a single type of labelleddeoxyribonucleoside triphosphate is attached to a primer molecule and

d) determining one or several nucleic acid sequences by means of thesequence-specific incorporation of the labelled deoxyribonucleosidetriphosphates.

In the method according to the invention at least two labelleddeoxyribonucleoside triphosphates are used in each case in a reactionmixture which each contain different labelling groups and differentbases i.e. a maximum of four deoxyribonucleoside triphosphates(corresponding to the bases A, T, C and G) can be used each of whichcarries a different labelling group. The labelling groups can beradioactive or non-radioactive labels and it should be possible todetect them concurrently. Non-radioactive labelling groups are preferredin particular fluorescent labelling groups e.g. fluorescent dyes such asrhodamine, derivatives of rhodamine such as methylrhodamine, Texas red,.phycoerythrin, fluorescein and derivatives thereof, CY3, CY5, CY2, CY7,coumarin, infrared 40, MR 200, bodipy dyes (molecular probes), 1,N⁶-etheno modification groups for dNTPs (molecular probes), IRD 40 etc.Other suitable non-radioactive labelling groups are for example metals,magnetic labelling groups which can be detected by nuclear resonance orby a supra-conducting quantum interferometric detector (SQID) orphosphorescent dyes. Suitable excitation or/and detection systems can beused to determine the respective labels.

Examples of commercially available fluorescent labelled dNTP are forexample fluorescein-dUTP, fluorescein-dATP (Boehringer Mannheim,Pharmacia); Texas red-dCTP and dGTP (NEN-Dupont), CY5-dATP and dCTP aswell as CY3-dATP (Pharmacia).

It is expedient to use fluorescent labelling groups which can bedetected concurrently. Thus one can for example use two or severaldifferent laser systems to excite the fluorescent groups which arecoupled to appropriate detection systems. Thus for example fluoresceinlabelling groups can be detected in automated DNA sequencing systemswith an argon laser (emission wavelength=488 nm) and appropriatedetectors. A helium-neon laser (emission wavelength=594 nm) can forexample be used as a second laser with appropriate detectors to detectnucleic acids labelled with Texas red. Interferences between both lasersystems can be substantially suppressed by band pass filters. The dataare recorded by two detector instruments, one for each laser. On theother hand it is also possible to use combinations of fluorescent dyeswhich can be excited by a single laser system and separately determinedby different detection systems in each case.

The concentration of the labelled dNTPs during the labelling reaction ispreferably so low that at a given polymerase concentration essentiallyonly a single labelled dNTP can be coupled to a primer molecule. Thelabelled dNTPs are therefore preferably used at a concentration of0.05-5 μmol/liter, particularly preferably at a concentration of 0.1-2.5μmol/liter. At such a dNTP concentration the polymerase present in thereaction mixture can statistically only attach a single dNTP molecule toa primer. The polymerase concentration is preferably 1-20 U,particularly preferably 5-15 U per reaction mixture. It may be criticalto adhere to the above-mentioned concentration ranges especially whenusing several primer molecules simultaneously since when the dNTP or/andenzyme concentration is too high several differently labelled dNTP maybe attached to a single primer.

Furthermore the specificity of the incorporation of labelled dNTPs canbe influenced by the selection of the labelling molecule (e.g.fluorescein, Texas red etc.), by the selection of the dNTP (dUTP, dTTP,DATP, dCTP, dGTP etc.) or/and by the type of linkage between the dNTPand the labelling molecule (e.g. length of the spacer arm).

In order to enzymatically attach a dNTP to the primer molecule, apolymerase, preferably the Klenow fragment of the E. coli DNA polymeraseI, unmodified T7 DNA polymerase, modified T7 DNA polymerase (Sequenase),T4 DNA polymerase, Taq DNA polymerase, Bst DNA polymerase or reversetranscriptase is used in the method according to the invention. Of theseenzymes polymerases without a 3' exonuclease activity are preferred e.g.modified T7 polymerase.

In particular embodiments of the invention polymerases with a 3'exonuclease activity e.g. unmodified T7 polymerase may, however, bepreferred for example if the labelled deoxyribonucleoside triphosphateis inserted as the last base of the primer molecule (position -1) bysequential exonuclease and polymerase activities.

An advantage of the method according to the invention is its simplicitysince no labelled primer molecules are required. This is particularlysignificant for large scale sequencing projects such as the sequencingof the human genome or other genome projects. On the other hand labelledprimers can of course also be used if desired in order to optionallyobtain additional information about the nucleic acid sequence.Furthermore the sequencing reactions can be carried out with acombination of unlabelled and labelled (fluorescent dyes or of anothertype e.g. digoxigenin, biotin, metals etc.) primers in which case theprimer is preferably selected such that the internal label is in eachcase only attached to the unlabelled primer. The labelled primerspreferably carry labelling groups that are different to those of thelabelled dNTPs.

When nucleic acids are sequenced simultaneously using two or more primermolecules, the selection of the primer molecules is important. Thus theprimers must be followed by different nucleotides which are incorporatedas the next base when the primer is extended by the polymerase. In thismanner only one type of labelling group is attached in each case to onetype of primer molecule in the labelling reaction. The incorporation ofother labelling groups can be excluded by selecting the reactionconditions in the labelling reaction.

A preferred embodiment of the present invention concerns a method inwhich at least two primer molecules are used which bind to differentregions of the nucleic acid to be sequenced wherein the primer moleculesare selected such that in each case the next base to be attached attheir 3' end is different and wherein a labelled deoxyribonucleosidetriphosphate with a corresponding base is used for each primer moleculesuch that only one single different labelled deoxyribonucleosidetriphosphate is attached to each primer molecule. In the presentapplication the base at the 3' end of a primer molecule is referred toin each case as "position -1" for the sake of clarity. The next base tobe attached by the polymerase to the 3' end of the primer molecule isreferred to as "position +1" and the base after that is referred to as"position +2".

Further criteria should also be observed preferably when constructingthe primer molecules to increase the specificity of the labellingreaction. Thus in one reaction mixture one preferably uses only thoseprimer molecules in which the base which is to be attached next at the3' end of the primer (position +1) is not the same as the base that isto be attached next but one (position +2) at the 3' end of anotherprimer. This reduces the probability that a primer molecule is labelledwith two different labelling groups.

Furthermore it is preferred that primer molecules are used in which thebase at the 3' end of a primer molecule (position -1) is not the same asthe next base (position +1) to be attached to the 3' end of anotherprimer molecule. In this manner it is possible to avoid potential doublelabelling due to a 3' exonuclease activity of the polymerase enzymeused. If a polymerase without 3' exonuclease activity is used e.g.modified T7 polymerase it is not absolutely necessary to follow thisselection criterium.

If two or several primer molecules are used it is possible tosimultaneously determine two or several nucleic acid sequences by themethod according to the invention.

The sequencing reaction of the method according to the invention isusually carried out in two steps. Firstly a single type of labelled dNTPis attached to the 3' end of the primer in a labelling reaction. It isparticularly preferred that only a single labelled dNTP is attached to aprimer in each case. The primer is preferably selected such that thelabelled dNTP can be attached to its 3' end (position +1). Furthermoreit is preferred that in the labelling reaction only labelled dNTP arepresent since, if there is an undesired binding of the primer tosecondary binding sites on the template, this reduces the probability ofincorporating labelled dNTPs. Finally the use of a DNA polymerasewithout 3' exonuclease activity e.g. Sequenase® is also preferredespecially when a primer ends with a nucleotide which is present as alabelled dNTP in the labelling reaction mixture.

As a next step an extension reaction takes place until a chaintermination molecule is incorporated which terminates thepolymerization. This extension reaction is preferably carried out inseparate reaction mixtures in the presence of the oligonucleotide primerthat is specifically labelled by the labelling reaction, the polymerase,the nucleic acid to be sequenced as a template, the four unlabelleddeoxyribonucleoside triphosphates and in each case one chain terminationmolecule. In this process a nucleic acid strand is formed by extensionof a primer which carries a marker group which is specific for therespective primer and which is terminated by the incorporation of achain termination molecule.

Hence the nucleic acid sequence is preferably determined by the methodaccording to the invention by

(d1) dividing the reaction mixture, after attaching the labelleddeoxyribonucleoside triphosphate, into several mixtures, adding anextension reagent to each mixture which contains four unlabelleddeoxyribonucleoside triphosphates and in each case a different chaintermination molecule and incubating the mixtures,

(d2) separating the nucleic acid fragments resulting from (d1) accordingto size and

(d3) determining the nucleic acid sequence by means of the labelling ofthe individual fragments.

In the extension reaction the four unlabelled dNTPs are in a largeexcess compared to the labelled dNTP used in the labelling reaction sothat no further incorporation of labelled dNTP takes place during theextension reaction. The unlabelled deoxyribonucleoside triphosphates arepreferably in an at least 100-fold excess, preferably in an at least1000-fold excess relative to the labelled deoxyribonucleosidetriphosphates. Therefore the concentration of the unlabelled dNTPs ispreferably 0.05-5 mmol/liter and particularly preferably 0.1-2.5 mmol/lliter in the extension mixture.

It is expedient to use deoxyribonucleoside triphosphates as chaintermination molecules which are modified at the 3' position of thedeoxyribose in such a way that they have no free OH group but arenevertheless accepted as a substrate by the polymerase. Examples of suchchain termination molecules are 3' fluoro, 3'-O-alkyl and 3'H-modifieddeoxyribonucleosides. 3'-H-modified deoxyribonucleotides are preferablyused as chain termination molecules i.e. dideoxyribonucleosidetriphosphates (ddNTP). It is preferable to use unlabelled chaintermination molecules in the method according to the invention but it isalso possible to use labelled chain termination molecules as known to aperson skilled in the art.

The determination of the nucleic acid sequence by means of the label isusually carried out by separating the labelled nucleic acid fragmentsaccording to length. This separation can be carried out according to allmethods known in the state of the art e.g. by various electrophoretic(e.g. polyacrylamide gel electrophoresis) or chromatographic (e.g. HPLC)methods, a gel electrophoretic separation being preferred. Furthermorethe labelled nucleic acids can be separated in any desired manner i.e.manually, semiautomatically or automatically, but the use of anautomated sequencer is generally preferred. In this case the labellednucleic acids can be separated in ultrathin plate gels of 20-500 μmpreferably 100 μm thickness (see e.g. Stegemann et al., Methods in Mol.and Cell. Biol. 2 (1991), 182-184) or capillaries. However, the sequencecan also be determined in non-automated devices e.g. by a blottingmethod.

If only a very small amount of the nucleic acid to be sequenced isavailable for a sequence determination according to the inventivemethod, an amplification step can also be carried out. One method ofamplification is to carry out one or several cycles of a polymerasechain reaction (PCR) using two primers before the actual sequencing. ThePCR is usually carried out without labelled dNTP. Thus the nucleic acidto be sequenced can be amplified before carrying out the actualsequencing procedure. On the other hand the amplification step can alsobe carried out with the aid of a thermocycling reaction. Thethermocycling reaction corresponds to a normal sequencing reactionwhich, however, is carried out in several cycles like a PCR. Thereaction mixture contains the nucleic acid template, the primermolecules, the dNTPs and the appropriate chain termination molecules aswell as the polymerase which is preferably thermostable. In this mannera certain amount of the labelled nucleic acid fragments are alwayssynthesized per cycle and large amounts of labelled fragments can beproduced in several cycles.

The nucleic acid to be sequenced can be present in a single-stranded aswell as in a double-stranded form. Good results are obtained when thenucleic acid to be sequenced is located on a double-stranded DNA sectore.g. a plasmid, cosmid, bacteriophage (lambda or P1), a viral vector oran artificial chromosome.

A further subject matter of the invention is a reagent kit for theselective introduction of a single labelling group into a nucleic acidwhich, in addition to the other components necessary for the labellingor sequencing, contains at least two labelled deoxyribonucleosidetriphosphates which each contain different labelling groups anddifferent bases. The reagents can for example be present in the form ofa solution, a suspension or a lyophilisate. This reagent kit can be usedin particular to sequence nucleic acids and can contain additionalreagents (e.g. enzyme, primer, buffer solutions, unlabelled dNTPs,terminators).

It is intended to elucidate the invention by the following examples inconjunction with FIGS. 1-4.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a diagram of the sequencing procedure according to theinvention,

FIG. 2a and FIG. 2b shows the result of a plasmid sequencing withsimultaneous use of two unlabelled primers in the presence offluorescein-15-dATP and Texas red-5-dCTP,

FIG. 3 shows the result of an investigation of the several-foldincorporation of labelled dNTP and

FIG. 4 shows the result of an investigation regarding the incorporationof a label into the primer itself.

EXAMPLE 1 Simultaneous DNA Sequencing Using Two Primers

cDNA molecules derived from a human keratinocyte-cDNA bank were clonedaccording to standard methods into a Bluescript vector (Stratagene,LaJolla, Calif., USA). The plasmid DNAs were purified using Quiagen(Hilden, Germany) or Nucleobond AX ion exchanger columns(Macherey-Nagel, Duren, Germany).

All primers were developed using the computer programme gene skipper(EMBL, Heidelberg, Germany) or Oligo™, 4.1 (Medprobe, Oslo, Norway).Primer pairs were selected which do not have a tendency to form dimersand which had no further binding sites on the template or no tendency toform a hairpin structure. One of the primers was selected such that an"A" is attached as the next base to the 3' end. The second primer wasselected such that a "C" is attached as the next base. The primers weresynthesized on the multiple segment DNA synthesizer from EMBL (Ansorgeet al, Electrophoresis 13 (1992) 616-619).

The following reaction protocol was used for the sequencing.

1. Denaturing and Annealing

5-10 μg double-stranded DNA (5-8 kb) was mixed with 2 unlabelled primermolecules (in each case 2 pmol in a total volume of 12 μl) and denaturedby adding 1 μl 1 mol/1 liter NaOH solution and heating to 65° C. for 3minutes. After cooling for one minute to 37° C. and brieflycentrifuging, 1 μl 1 mol/liter HCl and 2 μl annealing buffer (1mol/liter Tris/HCl pH 8, 0.1 mol/liter MgCl₂) were added.

2. Labelling Reaction

In each case 1 μl 10 μmol/liter fluorescein-15-dATP, 10 μmol/liter Texasred-5-dCTP and either native T7 DNA polymerase (8 U/μl) or Sequenase (13U/μl) were added to the above mixture. The reaction mixture wasincubated for 10 min. at 37° C.

3. Extension and Termination

1 μl extension buffer (304 mmol/liter sodium citrate, 40 mmol/literMnCl₂, 324 mmol/liter dithiothreitol) was added to the mixture andmixed. Then the solution was divided into aliquots and added to 4separate extension/termination mixtures which were composed of 3 μl ofthe respective termination solutions (40 mmol/liter Tris/HCl pH 7.4, 50mmol/liter NaCl, 5 μmol/liter of the respective ddNTP, 1 mmol/liter eachof DATP, dCTP, C⁷ -dGTP and dTTP) and 1 μl DMSO. The reaction wasincubated for 5 min at 37° C. and terminated by addition of 4 μl stopsolution (6 mg/ml dextran blue and 20 mmol/liter EDTA, pH 7.3 indeionised formamide). Then the samples were denatured for 4 min at 85°C. and applied to a sequencing gel.

4. DNA Sequencer

The structure of the modified DNA sequencer is described in detail byWiemann et al (Anal. Biochem. 224 (1995), 117-121). In addition to theusual argon laser detector system, a helium neon laser with acorresponding detector was also mounted in the sequencing device. The Arlaser excites the fluorescein-labelled samples whereas the HeNe laser isused to excite Texas red-labelled DNA fragments. Both laser rays arepassed into the gel with the aid of a single light coupling plate whichis arranged between two spacers on one side of the gel. The spatialdistance of 0.7 cm between the lasers and the combination of twodifferent lasers, detector and filter systems with correspondingfluorophores ensures that there is no cross-detection of the sequencesignals.

5. Gel Electrophoresis

The DNA fragments were separated on denaturing 6.5% Duracyl (Millipore,Bedford, Massachusetts, USA) sequencing gels. The separation was carriedout over a distance of 18.5 cm in the case of the Texas red-labelledfragments and of 19.2 cm in the case of the fluorescein-labelledfragments using standard A.L.F. (Pharmacia, Uppsala, Sweden) glassplates. The sequences of both reactions were detected on-line and storedin a computer.

FIG. 1 shows a diagram of the principle of the simultaneous sequencingof nucleic acids. After denaturation two primers bind to theirrespective complementary regions on the nucleic acid template to besequenced. The templates can be derived from the same strand of asingle-stranded or double-stranded DNA or from different strands of thesame double-stranded DNA or from different DNAs. Two differentlylabelled dNTPs are used in the sequencing reaction. In the labellingstep of the sequencing reaction only the two labelled nucleotidesfluorescein-15-dATP and Texas red-5-dCTP are present.

A selective labelling of specific products with only one dye is achievedby incorporating the respective nucleotide as the first base directlydownstream of the primer molecule. This reaction is comparable to theminisequencing (Syvanen et al, Genomics 8 (1990), 684-692) of pointmutations in which bases attached directly to the primer are the factorwhich distinguishes whether a labelled dNTP is incorporated or notincorporated. The primers are selected such that the bases which areattached directly to the primers are different e.g. an "A" and a "C". Inthis labelling step only the correct nucleotide is always incorporatedinto each primer. After incorporation of the first labelled dNTP thepolymerase can have a break since the labelled dNTPs are present only ata low concentration. The polymerase could also fall from the templateand be available to extend further primer molecules up to theincorporation of the labelled dNTPs (e.g. in cycle sequencing).

No further labelled dNTP is incorporated in the extension andtermination reaction due to a large excess of unlabelled dNTP.

FIG. 2 shows sequence data that were obtained in a simultaneoussequencing on both strands of a plasmid DNA. Two unlabelled walkingprimers were used. FIG. 2a shows the sequence generated withfluorescein-15-dATP and FIG. 2b shows the sequence generated with Texasred-5-dCTP.

FIG. 3 shows the result of a test for multiple incorporation of labelleddNTP. Sequencing primers were selected with which firstly a "C" and thenan "A" or firstly an "A" and then a "C" are incorporated at positions +1and +2. The sequencing reactions were carried out with native T7 DNApolymerase. FIG. 3a shows the Texas red signals of a primer with a "C"at position +1 and an "A" at position +2. FIG. 3b shows the fluoresceinsignals of the same primer. FIG. 3c shows Texas red signals of a primerwith "A" at position +1 and a "C" at position +2. FIG. 3d showsfluorescein signals with the primer from FIG. 3c.

It can be seen in FIG. 3 that there was no incorporation offluorescein-15-dATP at position +2 (FIG. 3b). In contrast FIG. 3c showsthat there was a slight incorporation of Texas red-5-dCTP at position+2. Since labelling at pos. +2 is considerably weaker than the labellingat pos. +1, the sequence data obtained with the second primer are,however, also unequivocal.

FIG. 4 shows the results of a test for the incorporation of a labelwithin the primer. Sequencing primers are selected which contain an "A"as the 3' terminal base (position -1) and which are intended toincorporate a "C" as the next base (position +1) as well as primers withthe reverse base sequence. The sequencing reactions were either carriedout with native T7 DNA polymerase (n-T7) or Sequenase (Seq). FIG. 4ashows Texas red signals from a sequencing with Sequenase and a primerwith "A" as the 3' terminal base and a "C" at position +1. FIG. 4b showsfluorescein signals from a sequencing with Sequenase and with the primerfrom FIG. 4a. FIG. 4c shows Texas red signals from a sequencing withnative T7 polymerase and the primer of FIG. 4a. FIG. 4d showsfluorescein signals from a sequencing with native T7 DNA polymerase andthe primer of FIG. 4a. FIG. 4e shows fluorescein signals of a sequencingwith Sequenase and a primer with a "C" as the 3' terminal base and an"A" at position +1. FIG. 4f shows Texas red signals from a sequencingwith Sequenase and the primer from FIG. 4e.

When the reaction was carried out with Sequenase (version 2.0) and theprimer with a 3' terminal "A", only Texas red dCTP was incorporated atposition +1 (FIG. 4a) whereas fluorescein dATP at position -1 was notincorporated into the primer (FIG. 4b). In contrast using native T7 DNApolymerase a slight incorporation of fluorescein-dATP was seen withinthe primer (position -1) (FIG. 4d).

With a "C" as the last base of the primer and an "A" as the subsequentbase it was found that Texas red dCTP was incorporated at position -1even when using Sequenase (FIG. 4f). However, due to the low signalstrength there was no significant interference of the sequencingreaction.

We claim:
 1. A method for the simultaneous and specific labeling ofnucleic acids comprising:a) providing an enzymatic labelling reaction ina single reaction vessel, said reaction vessel comprising:i) one or moretarget nucleic acid molecules; ii) at least one nucleic acid primermolecule of a distinct sequence, wherein each primer molecule hybridizesto a distinct area of the target molecule or molecules; and iii) a firstlabelled 3' unblocked deoxyribonucleotide triphosphate of a particularbase, and a second labelled 3' unblocked deoxyribonucleotidetriphosphate of a different base, wherein the first labelleddeoxyribonucleotide triphosphate contains a different label than thesecond deoxyribonucleotide triphosphate; b) hybridizing at least oneprimer molecule to the target molecule or molecules; c) attaching atleast one of the labeled deoxyribonucleoside triphosphates to at leastone primer molecule; and d) detecting the labeled deoxyribonucleotidetriphosphates that are now attached to at least one primer molecule. 2.The method of claim 1 comprising:a) providing an enzymatic labellingreaction in a single reaction vessel, said reaction vessel comprising:i)one or more target nucleic acid molecules; ii) a first nucleic acidprimer molecule of a distinct sequence, and a second nucleic acid primermolecule of a second distinct sequence, wherein the first primermolecule and the second primer molecule hybridize to different anddistinct areas of the target molecule or molecules; and iii) a firstlabelled 3' unblocked deoxyribonucleotide triphosphate of a particularbase, and a second labelled 3' unblocked deoxyribonucleotidetriphosphate of a different base, wherein the first labelleddeoxyribonucleotide triphosphate contains a different label than thesecond deoxyribonucleotide triphosphate; b) hybridizing the first andsecond primer molecules to the target molecule or molecules; c)attaching the labeled deoxyribonucleoside triphosphates to the first andsecond primer molecules, under conditions in which the first labelleddeoxyribonucleoside triphosphate is attached to the first primermolecule, and the second labeled deoxyribonucleoside triphosphate isattached to the second primer molecule; and d) detecting the labeleddeoxyribonucleotide triphosphates that are now attached to the first andsecond primer molecules.
 3. The method of claim 1 wherein the labeled 3'unblocked deoxyribonucleoside triphosphate is attached directly to the3' end of each primer molecule at position +1.
 4. The method of claim 1wherein a DNA polymerase without 3' exonuclease activity is used toattach the labeled 3' unblocked deoxyribonucleotide triphosphates to theprimer molecules.
 5. The method of claim 4 wherein a modified T7 DNApolymerase is used.
 6. The method of claim 1 wherein a DNA polymerasewith 3' exonuclease activity is used to attach the labeled 3' unblockeddeoxyribonucleoside triphosphates to the primer molecules.
 7. The methodof claim 1 wherein the labeled 3' unblocked deoxyribonucleosidetriphosphate is in inserted in a substitution reaction as the last baseof the primer molecule at position -1, by sequential exonuclease andpolymerase activities of the DNA polymerase.
 8. The method of claim 1wherein the primer molecules do not contain labeling groups.
 9. Themethod of claim 1 wherein the primer molecules do contain labelinggroups, and the labeling groups on the primer molecules are not the sameas the labeling groups on the first or second labeled 3' unblockeddeoxyribonucleoside triphosphates.
 10. The method of claim 1 wherein thelabeling group on the primer molecules are selected from the groupconsisting of fluorescent dyes, metals, biotin and digoxigenin.
 11. Themethod of claim 1 wherein the labeled 3' unblocked deoxyribonucleosidetriphosphates are used at a concentration of 0.05 to 5 μmol/liter. 12.The method of claim 11 wherein the labeled 3' unblockeddeoxyribonucleotide triphosphates are used at a concentration of 0.1 to2.5 μmol/liter.
 13. The method of claim 1 wherein two different Drimermolecules are used and wherein the primer molecules are selected in sucha way that the base to be attached at the 3' end of the first primermolecule is different from the base to be attached at the 3' end of thesecond primer molecule.
 14. The method of claim 1 wherein two differentprimer molecules are used and wherein the base to be attached at the 3'end of the first primer molecule at position +1 is not the same as thebase that could be attached as position +2 of the second primermolecule.
 15. The method of claim 1 wherein two different Drimermolecules are used and wherein the base to be attached at the 3' end ofthe first primer molecule at position +1 is not the same as the basethat could be attached at position +1 of the second primer molecule. 16.The method of claim 1 wherein the first and the second 3' unblockeddeoxyribonucleoside triphosphates contain different fluorescent labelinggroups.
 17. The method of claim 16 wherein the fluorescent labelinggroups are selected from the group consisting of fluorescein orderivatives thereof, Texas red, rhodamine or derivatives thereof,phycoerythrin, CY3, CY5, CY2, CY7, coumarin, infrared 40, MR 200, bodipydyes, IRD 40 and 1,N⁶ -ethano modification groups.
 18. The method ofclaim 16 wherein the different fluorescent labeling groups can bedetected simultaneously.
 19. The method of claim 16 wherein two or moredifferent lasers are used for detection.
 20. The method of claim 16wherein a laser is used for detection.
 21. A method for the simultaneoussequencing of nucleic acids comprising:a) providing an enzymaticlabelling reaction in a single reaction vessel, said reaction vesselcomprising:i) one or more target nucleotide molecules; ii) a firstnucleic acid primer molecule of a distinct sequence, and a secondnucleic acid primer molecule of a second distinct sequence, wherein thefirst primer molecule and the second primer molecule hybridize todifferent and distinct areas of the target molecule or molecules; andiii) a first labelled deoxyribonucleotide triphosphate of a particularbase, and a second labelled deoxyribonucleotide triphosphate of adifferent base, wherein the first labelled deoxyribonucleotidetriphosphate contains a different label than the seconddeoxyribonucleotide triphosphate; b) hybridizing the first and secondprimer molecules to the target molecule or molecules; c) attaching thelabeled deoxyribonucleoside triphosphates to the first and second primermolecules, under conditions in which the first labelleddeoxyribonucleoside triphosphate is attached to the first primermolecule, and the second labeled deoxyribonucleoside triphosphate isattached to the second primer molecule; d) dividing the reaction mixturefrom the single vessel into separate vessels, and adding an extensionreagent, wherein each vessel contains four unlabeled deoxyribonucleosidetriphosphates and, in each separate vessel, a different chaintermination molecule, e) separating the nucleic acid fragments formed ind); and f) detecting the labels on the first and seconddeoxyribonucleotide triphosphates and determining the sequence of thetarget molecules.
 22. The method of claim 21 wherein thedeoxyribonucleoside triphosphates are used at a concentration of 0.05-5mmol/liter.
 23. The method of claim 22 wherein the deoxyribonucleosidetriphosphates are used at a concentration of 0.1-2.5 mmol/liter.